A major challenge of cancer therapy is preferential destruction of malignant cells with sparing of normal tissue. Critical for successful eradication of malignant disease are early detection and selective ablation of the malignancy. Photodynamic therapy (PDT) is a clinically effective and still evolving locally selective therapy for cancers. The utility of PDT has been demonstrated with various photosensitizers for multiple types of disease. It is FDA approved for early and late stage lung cancer, obstructive esophageal cancer, high-grade dysplasia associated with Barrett's esophagus, age-related macular degeneration and actinic keratoses. PDT employs tumor localizing photosensitizers that produce reactive singlet oxygen upon absorption of light which is believed to be responsible for the destruction of the tumor. Subsequent oxidation-reduction reactions also can produce superoxide anions, hydrogen peroxide and hydroxyl radicals which contribute to tumor ablation4. Photosensitizers have been designed which localize relatively specifically to certain subcellular structures such as mitochondria, which are highly sensitive targets. On the tumor tissue level, direct photodynamic tumor cell kill, destruction of the tumor supporting vasculature and possibly activation of the innate and adaptive anti-tumor immune system interact to destroy the malignant tissue6. The preferential killing of the targeted cells (e.g. tumor), rather than adjacent normal tissues, is essential for PDT, and the preferential target damage achieved in clinical applications is a major driving force behind the use of the modality. The success of PDT relies on development of tumor-avid molecules that are preferentially retained in malignant cells but cleared from normal tissues.
In efforts to develop effective photosensitizers with the required photophysical characteristics, compounds having a tetrapyrollic core ring were used. Usually, chlorophyll-a and bacteriochlorophyll-a were used as intermediates in synthesis. Extensive QSAR studies on a series of the alkyl ether derivatives of pyropheophorbide-a (660 nm) led to selection of HPPH (hexyl ether derivative), now in promising Phase II clinical trials. Photosensitizer development now extends to purpurinimide (700 nm) and bacteriopurpurinimde (780-800 nm) series with high singlet oxygen producing capability. Long wavelength absorption is important for treating large deep seated tumors, because longer wavelength light increases penetration and minimizes the number of optical fibers needed for light delivery within the tumor.
Various efforts have been made to target tumor cells so that an agent may destroy the tumor cells while sparing normal cells. Such systems are reliant upon specific receptors and as such must reach receptor location. This is a disadvantage since even though the agent may reach the targeted cell, it may not be effective unless the particular receptor is reached and bound.
Multiple, complementary techniques for tumor detection, including magnetic resonance, scintigraphic and optical imaging are under active development. Each approach has particular strengths and advantages. Optical imaging includes measurement of absorption of endogenous molecules (e.g. hemoglobin) or administered dyes, detection of bioluminescence in preclinical models, and detection of fluorescence from endogenous fluorophores or from targeted exogenous molecules.
Fluorescence imaging is a non-invasive and non-ionizing imaging technique that requires only nanomoles of fluorophores for contrast enhancement. The NIR spectral range (˜650-950 nm) is known as the “biological window” for optical imaging since light absorption due to water, deoxygenated hemoglobin and oxygenated hemoglobin is minimized in this region, as well as tissue autofluorescence and light scattering.
Fluorescence, the emission of absorbed light at a longer wavelength, can be highly sensitive: a typical cyanine dye with a lifetime of 0.6 nsec can emit up to 1032 photons/second/mole. A sensitive optical detector can image<103 photons/second. Thus even with low excitation power, low levels of fluorescent molecular beacons can be detected. A challenge is to deliver the dyes selectively and in high enough concentration to detect small tumors. Use of ICG alone to image hypervascular or “leaky” angiogenic vessels around tumors has been disappointing, due to its limited intrinsic tumor selectivity. Multiple approaches have been employed to improve optical probe-localization, including administering it in a quenched form that is activated within tumors, or coupling it to antibodies or small molecules such as receptor ligands. Recent studies have focused on developing dye conjugates of small bioactive molecules, to improve rapid diffusion to target tissue and use combinatorial and high throughput strategies to identify, optimize, and enhance in vivo stability of the new probes. Some peptide analogs of ICG derivatives have moderate tumor specificity and are entering pre-clinical studies. However, none of these compounds are designed for both tumor detection and therapy. It is important to develop targeting strategies that cope with the heterogeneity of tumors in vivo, where there are inconsistent and varying expressions of targetable sites.
Photosensitizers (photosensitizer) generally fluoresce and their fluorescence properties in vivo has been exploited for the detection of early-stage cancers in the lung, bladder and other sites. For treatment of early disease or for deep seated tumors the fluorescence can be used to guide the activating light. However, photosensitizer are not optimal fluorophores for tumor detection for several reasons: (i) They have low fluorescence quantum yields (especially the long wavelength photosensitizers related to bacteriochlorins). Efficient photosensitizer tend to have lower fluorescence efficiency (quantum yield) than compounds designed to be fluorophores, such as cyanine dyes because the excited singlet state energy emitted as fluorescence is instead transferred to the triplet state and then to molecular oxygen. (ii) They have small Stokes shifts. Porphyrin-based photosensitizer have a relatively small difference between the long wavelength absorption band and the fluorescence wavelength (Stokes shift), which makes it technically difficult to separate the fluorescence from the excitation wavelength. (iii) Most photosensitizers have relatively short fluorescent wavelengths, <800 nm, which are not optimal for detection deep in tissues.
Attempts have been made to develop bifunctional conjugates that use tumor-avid photosensitizer to target the NIR fluorophores to the tumor. The function of the fluorophore is to visualize the tumor location and treatment site. The presence of the photosensitizer allows subsequent tumor ablation. The optical imaging allows the clinician performing PDT to continuously acquire and display patient data in real-time. This “see and treat” approach may determine where to treat superficial carcinomas and how to reach deep-seated tumors in sites such as the breast, lung and brain with optical fibers delivering the photo-activating light. A similar approach was also used for developing potential PDT/MRI conjugates in which HPPH was conjugated with Gd(III)DTPA Due to a significant difference between imaging and therapeutic doses, the use of a single molecule that includes both modalities is problematic.
Positron emission tomography (PET) is a technique that permits non-invasive use of radioisotope labeled molecular imaging probes to image and assay biochemical processes at the level of cellular function in living subjects20. PET predominately has been used as a metabolic marker, without specific targeting to malignancies. Recently, there has been growing use of radiolabeled peptide ligands to target malignancies. Currently, PET is important in clinical care and is a critical component in biomedical research, supporting a wide range of applications, including studies of tumor hypoxia, apoptosis and angiogenesis21. For targeting, a long circulation time may be desirable, as it can increase delivery of the agent into tumors. HPPH and the iodobenzyl pheophorbide-a have plasma half lives ˜25 h. The long radiological half life of 124I is well matched to the pheophorbides; it permits sequential imaging with time for clearance from normal tissue. Labeling techniques with radioiodine are well defined with good yield and radiochemical purity22. Despite the complex decay scheme of 124I which results in only 25% abundance of positron (compared with 100% positron emission of 18F), in vivo quantitative imaging with 124I labeled antibodies has been successfully carried out under realistic conditions using a PET/CT scanner A variety of biomolecules have been labeled with 124I. We have devised a coupling reaction which rapidly and efficiently links 124I to a tumor-avid photosensitizer23-25, and used the conjugate to target and image murine breast tumor and its metastasis to lung Acquisition of clinical PET images can be slow, but combination PET-CT scanners allow real time guidance of therapeutic interventions. Also, new developments in tracking may permit real time interventions guided by PET data sets.
Both cancer detection and treatment depend on selective delivery of appropriate agents to the malignancy. Photodynamic therapy (PDT), a relatively new modality for the treatment of a variety of oncological, cardiovascular, dermatological and ophthalmic diseases, is based on the preferential localization of photosensitizing molecules, (photosensitizers, PS) in target tissues. Upon light activation, the PS produces reactive singlet oxygen5 which damages tumor cells and neovasculature, and also initiates antitumor inflammatory and immune responses. We and others have developed relatively tumor-avid PS which selectively accumulate in tumor, and these molecules have been used to carry optical, PET and MR imaging agents to the tumor sites. However, the tumor selectivity of current PS is not always adequate. Approaches that link PS to antibody fragments or receptor ligands have been disappointing because the number of required PS/cell generally is greater than the number of antigen or receptor binding sites. Conversely, the imaging agent carrying capacity of the individual PS molecules is limited.
It has recently been shown that HPPH, developed in our laboratory and currently under Phase I/II clinical trials, when conjugated with certain cyanine dyes can be used for both fluorescence imaging and photodynamic therapy. The conjugate showed potential tumor imaging and PDT efficacy, but compared to the imaging dose the required therapeutic dose was 8-fold higher. Increasing the number of HPPH moieties in synthetic photosensitizer-cyanine dye (PS-CD) conjugates did not minimize the therapeutic dose.
Nanoscience is being developed in conjunction with advanced medical science for further precision in diagnosis and treatment. Nanoplatforms and nanovectors that deliver a therapeutic or imaging agent for biomedical applications show promise for cancer diagnosis and therapy. Therapeutic examples include nanoparticles containing PDT agents, folate receptor-targeted, boron containing dendrimers for neutron capture and nanoparticle-directed thermal therapy.
Nanoparticles have had disadvantages when considered for use in photodynamic therapy (PDT). In particular, certain nanoparticles have no relatively large knowledge base on cancer imaging, PDT, chemical sensing, stability and biodegradation. (2) have in in-vivo toxicity. (3) Have short plasma circulation time without surface modification and unstable or uncontrollable biodegradation and bioelimination rates (4) Have problems associated with scale-up and are not storage stabile over extended periods. And (5) have additional limitations including relative difficulty in incorporating hydrophobic compounds, leaching of small hydrophilic components unless they are “anchored”, and unknown limitation on bulk tumor permeability because of hydrogel swelling.
Nevertheless, nanoparticles (NP) are uniquely promising in that (i) their hydrophilicity and charge can be altered; (ii) they possess enormous surface area which can be modified with functional groups possessing a diverse array of chemical and biochemical properties, including tumor-selective ligands; (iii) owing to their sub-cellular and sub-micron size, they can penetrate deep into tissues and are generally taken up efficiently by cells.